Immunomodulatory drug fingolimod (FTY720) restricts the growth of opportunistic yeast Candida albicans in vitro and in a mouse candidiasis model

Fingolimod (FTY720) is a drug derived from the fungicidal compound myriocin. As it was unclear whether FTY720 has antifungal effects as well, we aimed to characterize its effect on Candida albicans in vitro and in a mouse candidiasis model. First, antifungal susceptibility testing was performed in vitro. Then, a randomized, six-arm, parallel, open-label trial was conducted on 48 mice receiving oral FTY720 (0.3 mg/kg/day), intraperitoneal C. albicans inoculation, or placebo with different combinations and chorological patterns. The outcome measures of the trial included serum concentrations of interleukin-10 and interferon-gamma, absolute lymphocyte counts, and fungal burden values in the mice’s livers, kidneys, and vaginas. Broth microdilution assay revealed FTY720’s minimum inhibitory concentration (MIC99) to be 0.25 mg/mL for C. albicans. The infected mice treated with FTY720 showed lower fungal burden values than the ones not treated with FTY720 (p<0.05). As expected, the mice treated with FTY720 showed a less-inflammatory immune profile compared to the ones not treated with FTY720. We hypothesize that FTY720 synergizes the host’s innate immune functions by inducing the production of reactive oxygen species. Further studies are warranted to unveil the mechanistic explanations of our observations and clarify further aspects of repurposing FTY720 for clinical antifungal usage.

Response: Thank you for your recommendation. As mentioned previously, the experiments are no longer in progress, therefore, we are unfortunately limited in presenting standard photographic documentations. We truly hope our explicit and detailed disclosure of the experimental procedures according to the EQUATOR guidelines and encouragement of future replicative studies could at least, partially resolve your concerns about the reproducibility of the results. We are also, hoping that the publication of our study, if considered of merit based on novelty and relevance, leads to conduction of further studies on the subject accounting for our limitations (including provision of standard figures) and validating the results. Once again, we would like to appreciate your punctiliousness and explicitly in critically appraising our study, and thank you for your time and consideration.  The author(s) received no specific funding for this work.

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Introduction
42 Fingolimod (FTY720) is an immunomodulatory drug currently indicated and approved by the FDA for 43 treatment of the relapsing forms of multiple sclerosis (MS)an autoimmune entity involving the CNS. It is 44 used off-label for progressive MS and other autoimmune neuropathies, while being evaluated for extra-45 nervous pathologies as well (2-4). FTY720 is considered a prodrug of its phosphorylated form fingolimod-46 phosphate (FTY720-P). Being a modulator of the sphingosine-1-phosphate receptors (S1PR) by structural 47 analogy, FTY720-P induces internalization of S1PR from the surface of lymphocytes, hindering their 48 trafficking outside the primary lymphatics and, therefore, keeping them away from inflammatory sites in end 49 organs (1). 50 While being associated with beneficial outcomes and reasonable tolerability among people with immune-51 mediated conditions such as MS (2, 3), safety and toxicity studies have shown both dose-dependent and 52 time-dependent adverse effects associated with FTY720 (4). The time-dependent adverse effects have 53 been opportunistic infections, reactivation of latent viruses, malignancies, etc., and were mostly deemed to 54 be due to chronic immunosuppression (4). Among the dose-dependent adverse effects, the most common 55 has been cardiotoxicity (2, 3). S1PRs are highly expressed on cardiomyocytes; the initial agonistic effect of 56 FTY720-P on S1PRs before inducing their internalization seems to be responsible for its dose-dependent 57 cardiotoxic effects (5). Hepatotoxic dose-dependent effects have also been reported (2, 3, 6), deemed to 58 be associated with FTY720's metabolic burden on the liver (7). Other dose-dependent unwanted and toxic 59 effects of FTY720 due to S1PR agonism, e.g., macular edema (8), unwanted loss of weight and appetite 60 (9), etc. have been reported. 61 FTY720 itself was derived from myriocin (ISP-1)a secondary metabolite of the entomopathogenic fungus 62 Isaria sinclairii and a potent inhibitor of the serine palmitoyltransferase (SPT) enzyme in the de-novo 63 sphingolipid biosynthesis pathway (10). Myriocin alters a metabolic pathway in eukaryotic cells vital for their 64 stability and proliferation. Consistently, it has shown to have antifungal effects e.g., against Candida and 65 Aspergillus spp. (11,12). 66 While FTY720 has no activity against SPT, emerging studies (13, 14) are pointing towards S1PR-67 independent anti-proliferative effects of FTY720 in its non-phosphorylated form. Meanwhile, several cases 68 of opportunistic fungal infections are reported among people receiving FTY720 chronically (15,16). 69 Although these reports describe case studies and population-based evidence is lacking in this regard, it 70 seems unclear whether FTY720 facilitates fungal expansion in vivo by weakening the immune system, or 71 has retained the antifungal effect of its molecular ancestor. 72 The subject of identifying and confirming the possible antifungal properties of substances like FTY720 -73 which could be administered safely in humansgains more relevance when considering the rapid 74 emergence of serious mycosis cases resistant to our usual antifungal armamentarium. Among the 75 candidiasis cases, resistance to drugs is classically attributed to the less prevalent infections with non-76 full name 4 albicans species, however, recent studies are documenting an alarming increase in the antifungal 77 resistance of Candida (C.) albicansthe dominant pathogen in candidiasis cases (17,18). As the incidence 78 of opportunistic candidiasis is rising, and commensal C. albicans has a high prevalence (19), its resistance 79 to drugs could be a major complicating factor of the future candidiasis cases. 80 Therefore, we first aimed to determine whether FTY720 has antifungal effects in vitro using antifungal 81 susceptibility testing. Then, we aimed to characterize its effects on immunological responses and fungal 82 burden values in mice infected with the opportunistic yeast C. albicansone of the most prevalent causative 83 agents of systemic opportunistic mycosis. We hereby report the study in accordance with the Animal 84  146 For the in-vivo study, 48 female C57BL/6 mice (Royan Institute, Iran)each weighing 22 (±3) grams -147 were prepared in accordance with the National Research Council guidelines for laboratory animal care (22). 148

Preparation, allocation, and follow-up of the mice
All mice were kept for one week before randomization, in standard cages in a humidity-controlled room at 149 a 25 °C (±2) temperature and a 12 hours daylight, 12 hours darkness cycle. Thereafter, each mouse was 150 identification-marked by an ear tag, and allocated to each one of the study arms based on a random 151 sequence generated by the NumPy software package (for Python version 2.7 on MacOS). They received 152 their interventions in an open-label manner and were followed up daily for 21 days after starting oral 153 FTY720/placebo. FTY720 (0.3 mg/kg/day) or oral placebo was administered by gastric gavage in form of a 154 100 μL suspension. For determination of frequency and dosage of FTY720, we selected a regimen 155 frequently used in previous studies, proven to bear immunomodulatory effects, and associated with minimal 156 adverse reactions in C57BL/6 mice (23-29). The mice assigned to be infected with C. albicans were 157 intraperitoneally injected with an inoculum containing 1.5 x 10 5 colony forming units (CFU) of C. albicans, 158 while the others were injected with normal saline in the same amounts. At the end of the follow-up, the mice 159 were sedated with ketamine-xylazine (100-10 mg/kg) and 1 mL of blood was taken from each through 160 cardiac catheterization. The blood samples were taken in anticoagulated tubes, and were promptly sent to 161 be prepared for CBC diff and cytokine assays. Then, through sterile procedures, the mice's livers, kidneys, 162 and vaginal samples were extracted and sent to be prepared for fungal culture. The mice were, thereafter, 163 euthanized with anesthetic overdose per American Veterinary Medical Association (AVMA) guidelines (30). 164 165 In order to characterize the mice's immune responses, absolute WBC and lymphocyte counts, serum IL10 166 (an anti-inflammatory cytokine), and IFNγ (a proinflammatory cytokine) concentrations were measured. 167 CBC diff was performed on whole blood samples by manual hemocytometry (Neubauer chamber) (31). 168

Analyses of samples
IFNγ and IL10 concentrations were quantified using ELISA (BioAssay™ kit for mouse, USBiological, USA) 169 in accordance with the manufacturer's instructions. For fungal culture, the vaginal samples were directly 170 spread on SDA plates. The livers and kidneys were first homogenized and filtered (using sterile techniques 171 and equipment), were diluted in ten serial concentrations, and then spread on SDA plates. After a 30-hour 172 incubation at 37 °C, the number of CFU's were counted and documented. 173 Data preparation and determination of appropriate tests 175 The distributions of immune response measures (IFNγ, IL10. WBC, and ALC) were assumed to be 176 lognormal; these measures were log10-transformed for the analyses. The distribution of fungal burden 7 values was assumed to be normal. The normality/lognormality assumptions were tested using the 178 33); if the assumptions were confirmed by alpha>0.05, parametric tests 179 (e.g., one-way analysis of variance [ANOVA]), and if not, non-parametric tests (e.g., Kruskal-Wallis) were 180 used for those measures. Post-hoc and correction for multiple comparisons was performed by controlling 181 the false discovery rate, using the two-stage step-up method of Benjamini,Krieger,and Yakutielli (34). 182

Statistical methods
In order to determine whether a single pooled variance could be used in the parametric comparisons, the 183 group variances were compared using the . In cases of insignificant (P>0.05) 184 difference among variances of the arms, a single pooled variance was calculated and used; otherwise, the 185 Welch and Brown-Forsyth method (36) was used (variances were calculated for individual arms separately). 186

187
To confirm if the follow-up period was sufficient for FTY720 to implement its immunological effects, the 188 IFNγ, IL10, WBC, and ALC measures were compared between the control arm (arm 1) and the arm 189 receiving FTY720 with injectable placebo (arm 2). 190 To confirm if the follow-up period was sufficient for the mice to develop an immune response against the 191 fungal infection, the IFNγ, IL10, WBC, and ALC measures in the control arm (arm 1) were compared with 192 the arm receiving IP C. albicans inoculum with oral placebo (arm 3). 193 To confirm if the follow-up period was sufficient for C. albicans to disseminate in vivo, the fungal burden 194 values in the control arm (arm 1) were compared with the arm receiving IP C. albicans inoculum with oral 195 placebo (arm 3). 196 To characterize the effect of FTY720 on mice's immunological response to C. albicans, the IFNγ, IL10,197 WBC, and ALC measures in the arms 4,5,6 (i.e., the arms receiving both FTY720 and IP C. albicans) were 198 once compared with the arm receiving FTY720 with injectable placebo (arm 2), and once with the arm 199 receiving IP C. albicans with oral placebo (arm 3). 200 In order to characterize the effect of FTY720 on the fungal burden in vivo, the liver, kidney, and vaginal 201 colony count values in arms 4,5,6 (i.e., the arms receiving both FTY720 and IP C. albicans) were compared 202 with the arm receiving IP C. albicans with oral placebo (arm 3). 203

204
The Prism software (version 9.0.2 for MacOS; GraphPad Software LLC.) was used for statistical analysis 205 and graphing. 206 207 In order to ensure the reproducibility of the results, the Enhancing the QUAlity and Transparency Of health 208

Reproducibility, safety and ethical considerations
Research (EQUATOR) guidelines (37) were followed to report the study, enabling all researchers across 209 the globe to repeat the experiments, obtain, compare, and validate the results. 210 8 The laboratory researchers followed strict regulatory standards in terms of safety; microbiological Petri 211 dishes were not handled without gloves during any of the experiments, and the laboratory researchers wore 212 sterile gowns, protective face-shields and masks, as well as following standard hand-washing protocols 213 with povidone iodine solution before entering and after exiting the laboratory. All of the safety procedures 214 were approved by the regulatory scientific review board who were monitoring the experiments through their 215 surveillance systems. 216 The animal subjects of this study were kept in standard cages and were not restricted in terms of space, 217 water, and food. All procedures on animal subjects were executed in accordance with national ethical 218 guidelines. This study was approved by the ethics committee of the Islamic Azad University Falavarjan 219 branch (Approval ID: IR.IAU.FALA.REC.1401.010). 220

222
In the well diffusion assay, the lowest concentration of FTY720 for which a visible zone of inhibition 223 surrounded its corresponding well was 0.5 mg/mL (Fig.2), hence, the probable MIC of FTY720 was 224 estimated to be 0.5 mg/mL or lower. As mentioned, we then used the resulted qualitative measures for 225 precise and standard MIC measurement with a microbroth dilution assay, in which the MIC99 was measured 226 to be 0.25 mg/mL, and the MIC50 was measured to be 0.12 mg/mL (Fig.3). These results confirmed that 227  231 The study was conducted per protocol; no unintended events happened during the study. The mice were 232 randomized and allocated to the study arms two days before receiving their oral interventions (T -2). All of 233 the randomized mice completed were followed up for 23 days (until T +21), except for one mouse in the 234 control arm (arm 1), which died due to an unknown reason in the second day of oral normal saline 235 consumption (T +2, four days after randomization). An investigative dissection of the mouse was conducted 236 to identify the possible reason of death, but no gross abnormalities were detected. Samples from the normal 237 saline administered to the mouse was cultured, resulting in growth of no microorganisms after one week. 238

Overview and validation of the in-vivo study
Apart from the signs of sepsis in the mice receiving IP C. albicans inoculum, the other mice experienced 239 no serious adverse events during their follow-up. 240 At the end of the study, the immunological profile of the mice treated with FTY720 and injectable placebo 241 (arm 2) significantly differed from the controls (arm 1) (Fig.4a), indicating a sufficient follow-up period for 9 FTY720 to execute its immunomodulatory effects. FTY720 significantly decreased IFNγ, increased IL10, 243 and decreased white blood cell (WBC) and absolute lymphocyte (AL) counts (Fig.4a). 244 The C. albicans-infected mice receiving oral placebo (arm 3) showed significantly different immunological 245 profiles and fungal burden measures with the controls (arm 1) (Fig.4a); this indicated that the follow-up 246 period was sufficient for dissemination of C. albicans in vivo, and for an antifungal immunological response 247 to develop. Disseminated candidiasis significantly increased IFNγ, WBC, and AL counts but insignificantly 248 affected IL10 (Fig.4a). Cultures of liver, kidney, and vaginal samples of the mice not receiving IP C. albicans 249 showed no growth of fungi, whereas the samples from all of the C. albicans-injected ones showed growth 250 of C. albicansconfirmed with gross visual examination and direct smear microscopy ( Fig.5, Fig.6). In two 251 cultures (one kidney sample from a mouse in arm 3 and one vaginal sample from a mouse in arm 6) multiple 252 microorganisms were colonized, highly indicative of procedural contamination; they were excluded from 253 analyses. 254  258 In order to characterize the effect of FTY720 on the mice's immune response to C. albicans, we compared 259 the arms receiving the IP C. albicans inoculum before, after, or simultaneously with initiation of FTY720 260 (arms 4,5,6), with the ones receiving FTY720/injectable placebo or C. albicans/oral placebo (arms 2,3) 261 ( Fig.1). 262

Effect of FTY720 on mice's immune response to C. albicans
Similar to the mice receiving IP C. albicans/oral placebo (arm 3), C. albicans inoculation before, and 263 simultaneously with initiation of FTY720 (arms 4,5) significantly increased IFNγ levels compared to the mice 264 receiving FTY720/IP placebo (arm 2) ( Fig.4b) (eTable 1). However, C. albicans inoculation after two days 265 of FTY720 consumption (in arm 6) caused an insignificant increase in IFNγ levels; IFNγ levels were 266 significantly lower in these mice compared to the mice receiving C. albicans/oral placebo (arm 3) (Fig.4b) 267 (eTable 1). In simple words, being on FTY720 was required for at least two days at the time of candidiasis 268 induction, for it to hinder the IFNγ response of the mice to the disseminating fungal infection. 269 The mice infected before initiation of FTY720 (arm 4) had significantly lower levels of IL10 compared to the 270 ones receiving FTY720/IP placebo (arm 2), while being comparable to the ones receiving IP C. albicans/oral 271 placebo (arm3) (Fig.4b) (eTable 1). It could be interpreted that the prior fungal infection hindered the IL10-272 increasing effect of FTY720. Induction of candidiasis simultaneously with, or later than FTY720 initiation (in 273 arms 5,6) did not affect the IL10-increasing effect of FTY720 as shown in Figure 4b. 274 As interpreted before, FTY720 significantly decreased, and candidiasis significantly increased WBC and 275 AL counts in mice (Fig.4a). The mice which started taking FTY720 before or after candidiasis induction 276 10 (arms 4,6) showed significantly lower WBC and AL counts than the ones receiving IP C. albicans/oral 277 placebo (arm 2) (Fig.4b). Surprisingly, the mice with simultaneous initiation of FTY720 and induction of 278 candidiasis still showed significantly increased WBC and AL countssignificantly higher than the mice 279 receiving FTY720/injectable placebo (arm 2), and comparable to the ones receiving C. albicans/oral 280 placebo (arm 3) (Fig.4b) (eTable 1). 281

Effect of FTY720 on fungal burden in vivo 282
To demonstrate the antifungal effect of FTY720 in vivo, we cultured specimens of mice's livers, kidneys, 283 and vaginas 21 days after initiation of FTY720/oral placebo, and compared the resulted fungal burden 284 values between the mice receiving C. albicans/oral placebo (arm 3), and the ones receiving both FTY720 285 and IP C. albicans inoculum (arms 4,5,6). The mice receiving FTY720 before, after, or simultaneously with 286 induction of candidiasis showed significantly lower fungal burden measures in their livers and kidneys 287 (eTable 1) (Fig.7); interestingly, the ones initiating FTY720 and getting infected on the same day (arm 5) -288 which also showed the high AL counts beforehad the lowest liver/kidney fungal burdenssignificantly 289 lower than all of the other arms. Regarding the vaginal burdens, only the mice initiating FTY720 before 290 receiving the IP C. albicans inoculum (arm 6) showed lower values than the ones receiving IP C. 291 albicans/oral placebo (arm 3) (eTable 1) (Fig.7). 292 [ Figure 7 placeholder] 293

294
Our results demonstrated that FTY720 has antifungal effects against C. albicansboth in vitro and in vivo. 295 In the mice models of disseminated candidiasis, this effect varied based on the chronological relation of 296 FTY720 initiation and C. albicans inoculation. Our report is the first describing the in-vivo antifungal effect 297 of FTY720 as far as we know. 298 In line with our study, a recent ex-vivo study by Wei and colleagues pointed to the fungicidal effect of 299 FTY720, which synergized the effect of amphotericin B (38). Wei et al. showed that this fungicidal effect is 300 present against non-albicans Candida spp., Saccharomyces cerevisiae, and Cryptococcus neoformans as 301 well as C. albicans (38). They also showed that 0.011 mg/mL of FTY720 affects C. albicans' growth rate 302 similar to 0.08 mg/L of amphotericin B (38). In an attempt to characterize the cause of this effect, they 303 demonstrated that FTY720 induces production and hyperaccumulation of reactive oxygen species (ROS) 304 in a dose-dependent manner; scavenging of the ROS from the samples using N-acetyl cysteine (NAC) 305 compromised the fungicidal effect of FTY720 significantly. Therefore, Wei et al.'s study suggested 306 hyperaccumulation of ROS as the reason of FTY720's fungicidal effect (38). ROS accumulation in fungi 307 induced by unphosphorylated FTY720 has been observed in previous studies as well (14,39), the 308 mechanism of which is an area of active investigation. 309 Administration of FTY720 at 1.25 mg/day in healthy adultsabove which has proved unbeneficial for 310 people with MS (6)results in a maximum blood concentration (Cmax) of 10.2 (±2.7) ng/mL at steady-state 311 (7); this concentration is 25000 times lower than its MIC99 for C. albicans. Maximum concentrations of 312 FTY720 in kidney and liver tissues reach approximately 40 times the Cmax (40)still much less than its in-313 vitro MIC99. Therefore, the current FTY720 regimens in people with MS are doubted to directly restrict C. 314 albicans infection. In our in-vivo study, the mice received 0.3 mg/kg/day of FTY720. A single 0.3 mg/kg 315 dose of FTY720 is associated with a maximum liver and kidney concentration of around 1.5 μg/mL (40). 316 Considering a 10-fold steady-state concentration reached by daily administration, the maximum 317 concentration of FTY720 in liver and kidney reaches 15 μg/mL -17 times less than its in-vitro MIC99 and 8 318 times less than its MIC50. Despite this fact, we did observe the antifungal effect of FTY720 in vivo. Hence, 319 we speculate that FTY720 had a synergistic effect with host's innate immunity against C. albicans. The 320 yeast could antagonize oxidative stress up to a specific threshold by inducing antioxidant metabolic 321 pathways (41). The innate immunity is absent in vitro, therefore, higher concentrations of FTY720 are 322 needed to raise the ROS levels up to the threshold intolerable by the yeast. In vivo, the generated ROS 323 induced by FTY720, although may not be adequate by itself, is added by the ROS produced by the innate 324 immune cells, while also attracting more cells to the frontline (42) neoformans is similar to its effect on C. albicans, and we demonstrated that the in-vitro antifungal effect of 332 FTY720 against C. albicans is also present in vivo. The dose-dependency of FTY720's antifungal effect 333 may be the explanation behind this discrepancy. It could be hypothesized that FTY720's 334 immunosuppressive effect when administered chronically with low dosesas in people with MSfacilitates 335 fungal infections, as its antifungal effects are absent in concentrations reached with those regimens 336 (discussed above). 337 Another notable observation in our study was the mice with simultaneous C. albicans inoculation and 338 initiation of FTY720 (arm 5) had a prominent increase in AL counts, similar to the mice receiving C. 339 albicans/oral placebo (arm 3); however, their kidney and liver samples revealed significantly lower fungal 340 burdens. The decreased fungal burden values in these mice could not merely be explained by the increased 341 AL counts, otherwise, the mice receiving C. albicans/oral placebo (arm 3) who had similar AL counts would 342 have similarly shown low fungal burdens in their organs. Further fundamental work is warranted to explain 343 this interesting observation. 344 12 345 We showed that the immunomodulatory drug FTY720 has an antifungal effect in vitro, which is present at 346 even lower concentrations in vivo. The facilitation of ROS hyperaccumulation by FTY720which assists 347 the innate immune system in creating an antifungal environmentmay explain our observations. Future 348 studies are warranted to evaluate further aspects of repositioning FTY720 for antifungal use in clinical 349 settings. 350

10.
Data availability 376 All data produced in this study are presented within the manuscript, its figures, and supplemental contents. 377 Raw data is also available upon reasonable request from the corresponding author. 378

11.
References 379   Petri dishes were handled without gloves as seen in these photographs. a) Vaginal sample from a mouse 504 in arm 5; b) Liver sample from a mouse in arm 6; c) Vaginal sample from a mouse in arm 2 (above) and a 505 mouse in arm 3 (below); d) Kidney sample from a mouse in arm 3. 506    (2-4). FTY720 is considered a prodrug of its phosphorylated form fingolimod-46 phosphate (FTY720-P). Being a modulator of the sphingosine-1-phosphate receptors (S1PR) by structural analogy, FTY720-P induces internalization of S1PR from the surface of lymphocytes, hindering their 48 trafficking outside the primary lymphatics and, therefore, keeping them away from inflammatory sites in end 49 organs (1). 50 While being associated with beneficial outcomes and reasonable tolerability among people with immune-51 mediated conditions such as MS (2, 3), safety and toxicity studies have shown both dose-dependent and 52 time-dependent adverse effects associated with FTY720 (4). The time-dependent adverse effects have 53 been opportunistic infections, reactivation of latent viruses, malignancies, etc., and were mostly deemed to 54 be due to chronic immunosuppression (4). Among the dose-dependent adverse effects, the most common 55 has been cardiotoxicity (2, 3). S1PRs are highly expressed on cardiomyocytes; the initial agonistic effect of 56 FTY720-P on S1PRs before inducing their internalization seems to be responsible for its dose-dependent 57 cardiotoxic effects (5). Hepatotoxic dose-dependent effects have also been reported (2, 3, 6), deemed to 58 be associated with FTY720's metabolic burden on the liver (7). Other dose-dependent unwanted and toxic 59 effects of FTY720 due to S1PR agonism, e.g., macular edema (8), unwanted loss of weight and appetite 60 (9), etc. have been reported. 61 FTY720 itself was derived from myriocin (ISP-1)a secondary metabolite of the entomopathogenic fungus 62 Isaria sinclairii and a potent inhibitor of the serine palmitoyltransferase (SPT) enzyme in the de-novo 63 sphingolipid biosynthesis pathway (10). Myriocin alters a metabolic pathway in eukaryotic cells vital for their 64 stability and proliferation. Consistently, it has shown to have antifungal effects e.g., against Candida and 65 Aspergillus spp. (11,12). 66 While FTY720 has no activity against SPT, emerging studies (13, 14) are pointing towards S1PR-67 independent anti-proliferative effects of FTY720 in its non-phosphorylated form. Meanwhile, several cases 68 of opportunistic fungal infections are reported among people receiving FTY720 chronically (15,16). 69 Although these reports describe case studies and population-based evidence is lacking in this regard, it 70 seems unclear whether FTY720 facilitates fungal expansion in vivo by weakening the immune system, or 71 has retained the antifungal effect of its molecular ancestor. 72 The subject of identifying and confirming the possible antifungal properties of substances like FTY720 -73 which could be administered safely in in homo humansgains more relevance when considering the rapid 74 emergence of serious mycosis cases resistant to our usual antifungal armamentarium. Among the 75 candidiasis cases, resistance to drugs is classically attributed to the less prevalent infections with non-76 Commented [MOU4]: Reviewer 1 comment 5 toxicity related studies were incorporated, cited, and discussed in the manuscript per reviewer's request. was <1% and <50% were considered as the MIC99 and the MIC50, respectively. 145 146 For the in-vivo study, 48 female C57BL/6 mice (Royan Institute, Iran)each weighing 22 (±3) grams -147

Preparation, allocation, and follow-up of the mice
were prepared in accordance with the National Research Council guidelines for laboratory animal care (22). 148 All mice were kept for one week before randomization, in standard cages in a humidity-controlled room at 149 a 25 °C (±2) temperature and a 12 hours daylight, 12 hours darkness cycle. Thereafter, each mouse was 150 identification-marked by an ear tag, and allocated to each one of the study arms based on a random 151 sequence generated by the NumPy software package (for Python version 2.7 on MacOS). They received 152 their interventions in an open-label manner and were followed up daily for 21 days after starting oral 153 FTY720/placebo. FTY720 (0.3 mg/kg/day) or oral placebo was administered by gastric gavage in form of a 154 100 μL suspension. For determination of frequency and dosage of FTY720, we selected a regimen 155 frequently used in previous studies, proven to bear immunomodulatory effects, and associated with minimal 156 adverse reactions in C57BL/6 mice (23-29). The mice assigned to be infected with C. albicans were 157 intraperitoneally injected with an inoculum containing 1.5 x 10 5 colony forming units (CFU) of C. albicans, 158 while the others were injected with normal saline in the same amounts. At the end of the follow-up, the mice 159 were sedated with ketamine-xylazine (100-10 mg/kg) and 1 mL of blood was taken from each through 160 cardiac catheterization. The blood samples were taken in anticoagulated tubes, and were promptly sent to 161 be prepared for CBC diff and cytokine assays. Then, through sterile procedures, the mice's livers, kidneys, 162 and vaginal samples were extracted and sent to be prepared for fungal culture. The mice were, thereafter, 163 euthanized with anesthetic overdose per American Veterinary Medical Association (AVMA) guidelines (30). 164 165 In order to characterize the mice's immune responses, absolute WBC and lymphocyte counts, serum IL10 166 (an anti-inflammatory cytokine), and IFNγ (a proinflammatory cytokine) concentrations were measured. 167 CBC diff was performed on whole blood samples by manual hemocytometry (Neubauer chamber) (31). 168

Analyses of samples
IFNγ and IL10 concentrations were quantified using ELISA (BioAssay™ kit for mouse, USBiological, USA) 169 in accordance with the manufacturer's instructions. For fungal culture, the vaginal samples were directly 170 spread on SDA plates. The livers and kidneys were first homogenized and filtered (using sterile techniques 171 and equipment), were diluted in ten serial concentrations, and then spread on SDA plates. After a 30-hour 172 incubation at 37 °C, the number of CFU's were counted and documented. 173 174

Data preparation and determination of appropriate tests
175 The distributions of immune response measures (IFNγ, IL10. WBC, and ALC) were assumed to be 176 lognormal; these measures were log10-transformed for the analyses. The distribution of fungal burden 177 7 values was assumed to be normal. The normality/lognormality assumptions were tested using the 178 33); if the assumptions were confirmed by alpha>0.05, parametric tests 179 (e.g., one-way analysis of variance [ANOVA]), and if not, non-parametric tests (e.g., Kruskal-Wallis) were 180 used for those measures. Post-hoc and correction for multiple comparisons was performed by controlling 181 the false discovery rate, using the two-stage step-up method of Benjamini,Krieger,and Yakutielli (34). 182 In order to determine whether a single pooled variance could be used in the parametric comparisons, the 183 group variances were compared using the . In cases of insignificant (P>0.05) 184 difference among variances of the arms, a single pooled variance was calculated and used; otherwise, the 185 Welch and Brown-Forsyth method (36) was used (variances were calculated for individual arms separately). 186

Analyses and their rationale
187 To confirm if the follow-up period was sufficient for FTY720 to implement its immunological effects, the 188 IFNγ, IL10, WBC, and ALC measures were compared between the control arm (arm 1) and the arm 189 receiving FTY720 with injectable placebo (arm 2). 190 To confirm if the follow-up period was sufficient for the mice to develop an immune response against the 191 fungal infection, the IFNγ, IL10, WBC, and ALC measures in the control arm (arm 1) were compared with 192 the arm receiving IP C. albicans inoculum with oral placebo (arm 3). 193 To confirm if the follow-up period was sufficient for C. albicans to disseminate in vivo, the fungal burden 194 values in the control arm (arm 1) were compared with the arm receiving IP C. albicans inoculum with oral 195 placebo (arm 3). 196 To characterize the effect of FTY720 on mice's immunological response to C. albicans, the IFNγ, IL10,197 WBC, and ALC measures in the arms 4,5,6 (i.e., the arms receiving both FTY720 and IP C. albicans) were 198 once compared with the arm receiving FTY720 with injectable placebo (arm 2), and once with the arm 199 receiving IP C. albicans with oral placebo (arm 3). 200 In order to characterize the effect of FTY720 on the fungal burden in vivo, the liver, kidney, and vaginal 201 colony count values in arms 4,5,6 (i.e., the arms receiving both FTY720 and IP C. albicans) were compared 202 with the arm receiving IP C. albicans with oral placebo (arm 3). 203

8
The laboratory researchers followed strict regulatory standards in terms of safety; microbiological Petri 211 dishes were not handled without gloves during any of the experiments, and the laboratory researchers wore 212 sterile gowns, protective face-shields and masks, as well as following standard hand-washing protocols 213 with povidone iodine solution before entering and after exiting the laboratory. All of the safety procedures 214 were approved by the regulatory scientific review board who were monitoring the experiments through their 215 surveillance systems. 216 The animal subjects of this study were kept in standard cages and were not restricted in terms of space, 217 water, and food. All procedures on animal subjects were executed in accordance with national ethical 218 guidelines. This study was approved by the ethics committee of the Islamic Azad University Falavarjan 219 branch (Approval ID: IR.IAU.FALA.REC.1401.010). 220

222
In the well diffusion assay, the lowest concentration of FTY720 for which a visible zone of inhibition 223 surrounded its corresponding well was 0.5 mg/mL (eFig.21), hence, the probable MIC of FTY720 was 224 estimated to be 0.5 mg/mL or lower. As mentioned, we then used the resulted qualitative measures for 225 precise and standard MIC measurement with a microbroth dilution assay, in which the MIC99 was measured 226 to be 0.25 mg/mL, and the MIC50 was measured to be 0.12 mg/mL (Fig.32). These results confirmed that 227  232 The study was conducted per protocol; no unintended events happened during the study. The mice were 233 randomized and allocated to the study arms two days before receiving their oral interventions (T -2). All of 234 the randomized mice completed were followed up for 23 days (until T +21), except for one mouse in the 235 control arm (arm 1), which died due to an unknown reason in the second day of oral normal saline 236 consumption (T +2, four days after randomization). An investigative dissection of the mouse was conducted 237 to identify the possible reason of death, but no gross abnormalities were detected. Samples from the normal 238 saline administered to the mouse was cultured, resulting in growth of no microorganisms after one week. 239

Overview and validation of the in-vivo study
Apart from the signs of sepsis in the mice receiving IP C. albicans inoculum, the other mice experienced 240 no serious adverse events during their follow-up. 241 9 At the end of the study, the immunological profile of the mice treated with FTY720 and injectable placebo 242 (arm 2) significantly differed from the controls (arm 1) (Fig.43a), indicating a sufficient follow-up period for 243 FTY720 to execute its immunomodulatory effects. FTY720 significantly decreased IFNγ, increased IL10, 244 and decreased white blood cell (WBC) and absolute lymphocyte (AL) counts (Fig.43a). 245 The C. albicans-infected mice receiving oral placebo (arm 3) showed significantly different immunological 246 profiles and fungal burden measures with the controls (arm 1) (Fig.43a); this indicated that the follow-up 247 period was sufficient for dissemination of C. albicans in vivo, and for an antifungal immunological response 248 to develop. Disseminated candidiasis significantly increased IFNγ, WBC, and AL counts but insignificantly 249 affected IL10 (Fig.43a). Cultures of liver, kidney, and vaginal samples of the mice not receiving IP C.  259 In order to characterize the effect of FTY720 on the mice's immune response to C. albicans, we compared 260 the arms receiving the IP C. albicans inoculum before, after, or simultaneously with initiation of FTY720 261 (arms 4,5,6), with the ones receiving FTY720/injectable placebo or C. albicans/oral placebo (arms 2,3) 262 ( Fig.1). 263

Effect of FTY720 on mice's immune response to C. albicans
Similar to the mice receiving IP C. albicans/oral placebo (arm 3), C. albicans inoculation before, and 264 simultaneously with initiation of FTY720 (arms 4,5) significantly increased IFNγ levels compared to the mice 265 receiving FTY720/IP placebo (arm 2) ( Fig.43b) (eTable 1). However, C. albicans inoculation after two days 266 of FTY720 consumption (in arm 6) caused an insignificant increase in IFNγ levels; IFNγ levels were 267 significantly lower in these mice compared to the mice receiving C. albicans/oral placebo (arm 3) (Fig.43b) 268 (eTable 1). In simple words, being on FTY720 was required for at least two days at the time of candidiasis 269 induction, for it to hinder the IFNγ response of the mice to the disseminating fungal infection. 270 The mice infected before initiation of FTY720 (arm 4) had significantly lower levels of IL10 compared to the 271 ones receiving FTY720/IP placebo (arm 2), while being comparable to the ones receiving IP C. albicans/oral 272 placebo (arm3) (Fig.3b4b) (eTable 1). It could be interpreted that the prior fungal infection hindered the 273 IL10-increasing effect of FTY720. Induction of candidiasis simultaneously with, or later than FTY720 274 initiation (in arms 5,6) did not affect the IL10-increasing effect of FTY720 as shown in Figure 43b.  As interpreted before, FTY720 significantly decreased, and candidiasis significantly increased WBC and 276 AL counts in mice (Fig.43a). The mice which started taking FTY720 before or after candidiasis induction 277 (arms 4,6) showed significantly lower WBC and AL counts than the ones receiving IP C. albicans/oral 278 placebo (arm 2) (Fig.43b). Surprisingly, the mice with simultaneous initiation of FTY720 and induction of 279 candidiasis still showed significantly increased WBC and AL countssignificantly higher than the mice 280 receiving FTY720/injectable placebo (arm 2), and comparable to the ones receiving C. albicans/oral 281 placebo (arm 3) (Fig.43b) (eTable 1). 282 283 To demonstrate the antifungal effect of FTY720 in vivo, we cultured specimens of mice's livers, kidneys, 284 and vaginas 21 days after initiation of FTY720/oral placebo, and compared the resulted fungal burden 285 values between the mice receiving C. albicans/oral placebo (arm 3), and the ones receiving both FTY720 286 and IP C. albicans inoculum (arms 4,5,6). The mice receiving FTY720 before, after, or simultaneously with 287 induction of candidiasis showed significantly lower fungal burden measures in their livers and kidneys 288 (eTable 1) (Fig.74); interestingly, the ones initiating FTY720 and getting infected on the same day (arm 5) 289 which also showed the high AL counts beforehad the lowest liver/kidney fungal burdenssignificantly 290 lower than all of the other arms. Regarding the vaginal burdens, only the mice initiating FTY720 before 291 receiving the IP C. albicans inoculum (arm 6) showed lower values than the ones receiving IP C. 292 albicans/oral placebo (arm 3) (eTable 1) (Fig.74).

295
Our results demonstrated that FTY720 has antifungal effects against C. albicansboth in vitro and in vivo. 296 In the mice models of disseminated candidiasis, this effect varied based on the chronological relation of 297 FTY720 initiation and C. albicans inoculation. Our report is the first describing the in-vivo antifungal effect 298 of FTY720 as far as we know. 299 In line with our study, a recent ex-vivo study by Wei and colleagues pointed to the fungicidal effect of 300 FTY720, which synergized the effect of amphotericin B (38). Wei et al. showed that this fungicidal effect is 301 present against non-albicans Candida spp., Saccharomyces cerevisiae, and Cryptococcus neoformans as 302 well as C. albicans (38). They also showed that 0.011 mg/mL of FTY720 affects C. albicans' growth rate 303 similar to 0.08 mg/L of amphotericin B (38). In an attempt to characterize the cause of this effect, they 304 demonstrated that FTY720 induces production and hyperaccumulation of reactive oxygen species (ROS) 305 in a dose-dependent manner; scavenging of the ROS from the samples using N-acetyl cysteine (NAC) 306 compromised the fungicidal effect of FTY720 significantly. Therefore, Wei et al.'s study suggested 307 hyperaccumulation of ROS as the reason of FTY720's fungicidal effect (38). ROS accumulation in fungi 308   Petri dishes were handled without gloves as seen in these photographs. a) Vaginal sample from a mouse 505 in arm 5; b) Liver sample from a mouse in arm 6; c) Vaginal sample from a mouse in arm 2 (above) and a 506 mouse in arm 3 (below); d) Kidney sample from a mouse in arm 3. 507